Pediococcus acidilactici King01 for relieving oral malodor of pet dogs, microbial inoculum and application of microbial inoculum
By using the bacteria agent prepared by King01 of Lactate Tablets, the oral odor and dental health problems of pet dogs are solved, effectively inhibiting the occurrence of plaque, tartar and gingivitis, and significantly alleviating bad breath.
Patent Information
- Application Number
- CN202510213043.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-03-28
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to effectively alleviate oral odor in pet dogs, inhibit the formation of plaque and tartars, and reduce the occurrence of gingivitis. The conventional methods are cumbersome and costly.
King01, classified as Pediococcus acidilacticici, and preservation number is CGMCC No. 31635, is prepared as bacterial agent to inhibit the formation of plaque and tartars in canine dental plaque, reduce the occurrence of gingivitis, and relieve bad breath.
King01 of lactic acid tablets significantly reduces the concentration of volatile sulfides in the oral cavity of dogs, reduces breath, relieves bad breath, inhibits the formation of plaque and tartars, and reduces the occurrence of gingivitis.
Smart Images
Figure CN119709556A_ABST
Abstract
Description
Technical Field
[0001] The invention provides a Pediococcus acidilactici King01, a bacterial agent and an application thereof for alleviating bad breath in the mouth of a pet dog, and belongs to the technical field of microorganisms. Background Art
[0002] Bad breath is the most obvious oral problem for pet dogs. It is a sub-healthy condition that is widely found in the oral cavity of pets such as dogs and cats. Although bad breath is not a substantial disease, it may be the first sign of a serious oral disease and one of the first signs of periodontitis that pet owners notice. Bad breath has been with pets for a long time, which not only causes great damage to the health of pets, but also seriously affects the interaction between pets and humans. Pet bad breath is caused by the microenvironment in the oral cavity, mostly due to the production of volatile compounds. Currently known volatile compounds include sulfur-containing amino acids (such as cysteine and methionine), sulfur-containing compounds (such as methyl mercaptan, hydrogen sulfide and dimethyl sulfide), etc.
[0003] Bad breath in pet dogs and cats can be divided into pathological bad breath and physiological bad breath. Physiological bad breath is mostly short-term bad breath with little impact; while pathological bad breath is closely related to the environment in the oral cavity. For example, dental plaque, tartar, periodontitis, caries, poor restorations, residual roots, residual crowns, etc. can all cause bad breath in pets. When suffering from severe periodontal disease, there are a large number of bacteria in the periodontal pockets. These bacteria will produce the above-mentioned volatile sulfides during reproduction and metabolism, thus causing bad breath. Clinical correlation studies have shown that in studies on the correlation between periodontal disease and canine bad breath, the breath values of dogs with a large amount of dental plaque and tartar on the surface of their teeth are significantly higher than those of dogs with no visible plaque or tartar on their teeth. Therefore, without considering the existence of inherent periodontal disease, slowing down the deposition of dental plaque and tartar is of great significance in alleviating bad breath.
[0004] At present, the methods to relieve bad breath in pet dogs mainly include improving diet, brushing teeth regularly, using teeth grinding tools, and washing teeth. It takes a certain amount of time to adapt to improving diet, and not all pet dogs can accept new types of food. Regularly brushing pet dogs' teeth with a special toothbrush and toothpaste can reduce the formation of dental plaque and tartar, but some pet dogs may not be used to brushing teeth, and will resist or avoid it, which requires patient training. If the operation is improper, it may cause damage to the pet dog's mouth. Teeth grinding tools are only suitable for the occlusal end of the teeth and have no effect on the end of the teeth. Taking pet dogs to the pet hospital for professional teeth washing regularly can thoroughly clean the teeth and mouth, but teeth washing may cause certain stress to pet dogs, and it needs to be done regularly, which is costly. The above methods have their applicable conditions and potential disadvantages for relieving bad breath in pet dogs. Therefore, there is an urgent need for a healthy, effective, easy-to-operate, time-saving, labor-saving and cost-saving method.
[0005] CN116200307A discloses a probiotic composition for cat oral care and its application, wherein the probiotic composition comprises animal bifidobacterium and lactic acid bacteria, wherein the lactic acid bacteria is any one or more combinations of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri. Animal experiments have verified that the cat oral care probiotic composition (containing Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, animal bifidobacterium, Lactobacillus plantarum, with a total viable count of 1.8×10 10 After 14 days of uniform mixing of the probiotic composition with the toothpaste matrix, the cat's bad breath was resolved, and the sensory score index of bad breath was significantly reduced compared to the cat fed with the toothpaste without probiotics. However, the effect described is the effect of the probiotic composition and the toothpaste in combination, and the application does not report the effect that can be achieved when the probiotic composition is used alone or when a certain strain in the composition is used alone.
[0006] There is no report in the prior art that Pediococcus acidilactici is used alone to relieve bad breath in pet dogs' mouths, inhibit the formation of dental plaque and calculus, and reduce the occurrence of gingivitis. Summary of the invention
[0007] In view of the shortcomings of the prior art, the present invention provides a Pediococcus acidilactici King01, a bacterial agent and its application for alleviating bad breath in pet dogs, so as to achieve the following invention objectives: inhibit the formation of canine dental plaque and tartar, reduce the occurrence of gingivitis, and alleviate bad breath.
[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions: A Pediococcus acidilactici King01 for alleviating bad breath in pet dogs' mouths, wherein the Pediococcus acidilactici King01 is classified and named Pediococcus acidilactici Pediococcus acidilactici , the deposit number is CGMCC No.31635.
[0009] The bacterial agent is prepared using the Pediococcus acidilactici King01.
[0010] The number of bacteria in the bacterial agent is (4.8-5.2)×10 9 cfu / g.
[0011] The application of Pediococcus acidilactici King01 in preparing a product for alleviating bad breath in pet dogs.
[0012] The lactic acid Pediococcus King01 is classified and named as lactic acid Pediococcus Pediococcus acidilactici, Deposit date: August 7, 2024, Deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration, Deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Deposit number: CGMCC No.31635.
[0013] Compared with the prior art, the present invention achieves the following beneficial effects: The Pediococcus acidilactici King01 of the present invention can inhibit the formation of canine dental plaque and calculus, and reduce the occurrence of gingivitis; The Pediococcus acidilactici King01 of the present invention can significantly reduce the concentration of volatile sulfides in the oral cavity of dogs, reduce bad breath, and alleviate bad breath. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 is a bar graph showing the change in dental plaque index in Example 4; Figure 2 is a bar graph showing the change in dental calculus index in Example 4; Figure 3 is a bar graph showing changes in gingivitis index in Example 4; Figure 4 This is a bar graph showing the changes in the tone value in Example 4. DETAILED DESCRIPTION
[0015] The preparation method of MRS plate culture medium is as follows: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, and 15g of agar are dissolved in distilled water and the volume is fixed to 1L; after mixing the above raw materials, the pH is naturally adjusted, after stirring evenly, sterilize at 121°C and 0.1MPa for 20min, pour the sterilized culture medium into a plate, let it cool and set aside.
[0016] The preparation method of MRS liquid culture medium is as follows: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, and 0.2g of magnesium sulfate heptahydrate are dissolved in distilled water and the volume is fixed to 1L; after mixing the above raw materials, the pH is adjusted to 6.8, and after stirring evenly, sterilized at 121°C and 0.1MPa for 20min.
[0017] Example 1 Isolation and Identification of Strain 1. Strain screening and purification (1) Source of the strain: healthy dog feces, collected on May 13, 2023 at Yuanlin Road, Qilin District, Qujing City, Yunnan Province.
[0018] (2) Sample preparation: ① Take 10 mL of sterilized saline (0.85%) and place it in a sterile conical flask, then add 1 g of healthy dog feces from step (1), shake and set aside; ② Dilute the solution in step ① to prepare samples with different concentration gradients, which are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , numbered 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, and are for use.
[0019] (3) Cultivation of strains: Take 100 μL of solution No. 5, No. 6, No. 7, and No. 8, respectively, and spread it on the MRS plate culture medium using a spreader, and culture it at 37°C under anaerobic conditions for 48 h.
[0020] (4) Isolation and purification: Pick a single colony with a milky white, round, smooth and opaque morphology and neat edges and streak it into three zones on an MRS plate medium. After streaking, culture it at 37°C under anaerobic conditions for 48 h. Repeat the above operation for a total of three streakings to purify the pure strain. Add the pure strain to a glycerol tube and store it at -80°C.
[0021] 2. Identification The single colony obtained after separation and purification was sent for identification. The identification unit was China Industrial Microbiological Culture Collection Management Center. The detection method was FMIC-QO01-001-2015 Microbiological Detection Bacterial Multiphase Identification Detection Method. The identification result was: the strain was Pediococcus acidilactici Pediococcus acidilactici .
[0022] The identified strain was named Pediococcus acidilactici King01 and sent to the General Microbiological Center of China Microbiological Culture Collection Administration for preservation. The preservation information is as follows: Pediococcus acidilactici King01, classified as Pediococcus acidilactici Pediococcus acidilactici , Deposit date: August 7, 2024, Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Deposit number: CGMCC No.31635.
[0023] Example 2 Preparation of Pediococcus acidilactici King01 bacterial agent (1) Take the preserved Pediococcus acidilactici King01 and activate it on the MRS plate medium, pick a single colony and transfer it to 100 mL of MRS liquid medium, culture it at 37°C for 12 h to prepare the seed solution, then inoculate it into MRS liquid medium at a 1% (v / v) inoculation amount, and then culture it at 37°C for 24 h to obtain the bacterial solution; (2) After centrifuging the bacterial solution, the cells were collected, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a concentration of 2.0 × 10 10 cfu / mL of bacterial suspension, freeze-drying the bacterial suspension to obtain bacterial powder; (3) The bacterial powder was mixed with isomaltooligosaccharide to prepare Pediococcus acidilactici King01 bacterial agent. Isomaltooligosaccharide was purchased from Baolingbao Biotechnology Co., Ltd.
[0024] In this example, the number of bacteria in the prepared Pediococcus acidilactici King01 inoculum was 5.0×10 9 cfu / g.
[0025] Example 3 Preparation of Pediococcus acidilactici King01 bacterial agent (1) Take the preserved Pediococcus acidilactici King01 and activate it on the MRS plate medium, pick a single colony and transfer it to 100 mL of MRS liquid medium, culture it at 37°C for 12 h to prepare the seed solution, then inoculate it into MRS liquid medium at a 1% (v / v) inoculation amount, and then culture it at 37°C for 24 h to obtain the bacterial solution; (2) After centrifuging the bacterial solution, the cells were collected, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a concentration of 2.0 × 10 10 cfu / mL bacterial suspension; (3) The bacterial suspension was sterilized at 121°C for 30 min, and then freeze-dried. After drying, it was mixed with oligomaltose (purchased from Baolingbao Biotechnology Co., Ltd.) according to the number of live bacteria in the bacterial suspension to prepare a suspension containing 5.0×10 inactivated Pediococcus acidilactici King01 cells. 9 cfu / g, and no live bacteria were found in the solid preparation after testing.
[0026] Example 4: Improvement of canine dental plaque, calculus, gingivitis and halitosis by Pediococcus acidilactici King01 60 healthy, similar-sized adult hybrid dogs were selected from a pet breeding center in Weifang, Shandong Province. After uniform ultrasonic dental cleaning, they were randomly divided into 4 groups, 15 in each group, with random gender and age between 1 and 5 years old. Before the start of the experiment (day 0), all dogs were anesthetized intravenously in a pet hospital for oral examination. The heartbeat, pulse and respiration of the dogs were monitored during the process. If the heart rate or respiration was abnormal, emergency measures were taken in time. To ensure the accuracy of the results and reduce the influence of human factors, all oral examinations were completed by the same veterinarian. The severity of dental plaque, tartar and gingivitis in all dogs was scored one by one. The plaque scoring standard is shown in Table 1, the tartar scoring standard is shown in Table 2, and the gingivitis scoring standard is shown in Table 3. Sedation and anesthesia were removed after the oral examination.
[0027] In addition to the oral examination, the concentration of volatile sulfur compounds (VSCs) in the oral cavity of all dogs was measured using a Halimeter breath analyzer (PLUS model, INTERSCAN) before the start of the experiment (on day 0). During the test, the Halimeter breath analyzer was connected to a disposable straw, which was placed in the oral cavity, and the breath analyzer was started. The value of the breath analyzer was observed. When the value reached the maximum value and began to decrease, the maximum value was recorded, and the average of the three records was taken.
[0028] During the experiment, the dogs were scored for dental index every 15 days, and the scoring indexes of dental plaque, calculus, and gingivitis were tested, and the VSCs concentration was recorded.
[0029] During the test, all test dogs were fed the same dog food. In addition to the dog food, starting from the first day of the test, the test group 1 was fed with the lactic acid Pediococcus King01 bacterial agent prepared in Example 2 of the present invention at an addition amount of 2 g / d per dog, the test group 2 was fed with the lactic acid Pediococcus King01 bacterial agent prepared in Example 3 of the present invention at an addition amount of 2 g / d per dog, the comparative group was fed with the lactic acid Pediococcus JYPA-16 bacterial agent (CGMCC No. 23352) at an addition amount of 2 g / d per dog, and the control group was not treated in any way.
[0030] The preparation method of the Pediococcus acidilactici JYPA-16 bacterial agent is to take the preserved Pediococcus acidilactici JYPA-16 and prepare it according to the method of Example 2 of the present invention.
[0031] Table 1 Scoring criteria for oral health status of dogs (dental plaque)
[0032] Table 2 Scoring criteria for oral health status of dogs (dental calculus)
[0033] Table 3 Scoring criteria for oral health status of dogs (gingivitis)
[0034] (1) Improvement of canine dental plaque by Pediococcus acidilactici King01 The changes of dental plaque index of all tested dogs during the trial period were as follows Figure 1 As shown. After teeth cleaning, the dental plaque index of the dogs in the control group, test group 1, test group 2 and comparison group was 0, and there was no difference in the dental plaque index of the four groups before the start of the experiment. On the 15th, 30th, 45th and 60th days, the dental plaque index of the control group and the comparison group was significantly higher than that of the test group 1 and test group 2, and the dental plaque index of the control group and the comparison group increased rapidly during the experiment. On the 60th day, the dental plaque index of the control group and the comparison group had reached more than 2 points; while the dental plaque index of the test group 1 and the test group 2 increased slowly. On the 60th day, the dental plaque index of the test group 1 was only 1.13 points, and the dental plaque index of the test group 2 was only 1.53 points.
[0035] (2) Improvement of canine dental calculus with Pediococcus acidilactici King01 The changes of dental calculus index of all tested dogs during the experiment were as follows: Figure 2 As shown. In the first 45 days of the experiment, the tartar indexes of different groups were very close, presumably because the formation of tartar takes a certain amount of time. After 45 days, the tartar indexes of the control group, test group 2, and comparison group began to grow rapidly. On the 60th day, the tartar indexes of the control group, test group 2, and comparison group were significantly higher than those of test group 1. The tartar indexes of the control group, test group 2, and comparison group were 1.20 points, 0.93 points, and 1.13 points, respectively, while the tartar index of test group 1 was only 0.53 points. This indicates that Pediococcus acidilactici King01 helps to delay the formation of canine tartar and is beneficial to maintaining the health of canine oral cavity and teeth.
[0036] (3) Improvement of canine gingivitis with Pediococcus acidilactici King01 The changes of gingivitis index of all tested dogs during the trial period are as follows: Figure 3As shown. Since the experimental dogs had not maintained good oral hygiene before the start of the experiment, a lot of dental plaque and tartar accumulated, so after teeth washing, a small number of dogs still suffered from gingivitis. On day 0, the gingivitis index between different groups was very close. During the entire observation period of 60 days, although the dogs taking probiotics (experimental group 1, experimental group 2 and control group) also had repeated gingivitis, it was only a very small number. The comprehensive gingivitis index of experimental group 1, experimental group 2 and control group was below 0.2 during the entire observation period. The comprehensive gingivitis index of the control group has been increasing, and the number of dogs with gingivitis is relatively large. On the 60th day, the comprehensive gingivitis index of the control group has reached 0.6 points. It shows that taking probiotic preparations can effectively reduce the probability of dogs suffering from gingivitis and has a certain effect on the treatment and prevention of gingivitis.
[0037] However, compared with Experimental Group 1 and Experimental Group 2, the probability of dogs in the control group suffering from gingivitis was relatively higher, so the effect of Pediococcus acidilactici King01 in treating and preventing gingivitis was better.
[0038] (4) Improvement of canine bad breath with Pediococcus acidilactici King01 The breath values of all the test dogs changed during the experiment. Figure 4 As shown. On day 0, there was no significant difference in the breath values of the groups. From day 15 to day 60, the breath values of the control group were significantly higher than those of test groups 1 and 2. In the first 30 days, the breath values of test groups 1, 2 and the comparative group were relatively close. From day 45, the breath values of the comparative group were significantly higher than those of test groups 1 and 2.
[0039] On the 60th day, the breath values of the control group and the comparative group were significantly higher than those of the experimental group 1 and the experimental group 2. The breath values of the four groups were 304.07ppb for the control group, 273.33ppb for the comparative group, 178.13ppb for the experimental group 1, and 209.80ppb for the experimental group 2.
[0040] This indicates that Pediococcus acidilactici King01 is beneficial to maintaining dog's oral hygiene and fresh breath, and can significantly reduce the concentration of volatile sulfides in the dog's oral cavity, reduce bad breath, and relieve bad breath.
Claims
1. A Pediococcus acidilactici King01 for alleviating bad breath in pet dogs, characterized by: The lactic acid Pediococcus King01 is classified and named as lactic acid Pediococcus Pediococcus acidilactici The deposit number is CGMCC No.31635, the deposit date is August 7, 2024, the deposit unit is the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.
2. A bacterial agent prepared using the Pediococcus acidilactici King01 according to claim 1.
3. The bacterial agent according to claim 2, characterized in that: The number of bacteria in the bacterial agent is (4.8-5.2)×10 9 cfu / g.
4. Use of Pediococcus acidilactici King01 according to claim 1 in preparing a product for alleviating bad breath in pet dogs.